FINDING THE MOLECULAR DEFECT IN XERODERMA PIGMENTOSUM

遗传性疾病着色性干皮病（XP）的特征是严重的 对紫外线（UV）辐射的敏感性和皮肤的高发病率 癌 生物化学缺陷是在一个途径的切除修复 紫外线、抗肿瘤药物顺铂和许多其他 产生大量DNA加合物的试剂。 这种DNA修复途径涉及 多种蛋白质，因为至少有七种基因互补 A组至G组。修复的早期步骤必须包括认识到 DNA损伤的部位 最近，一种具有这种功能的候选蛋白， XPE结合因子（XPE-BF）是本实验室鉴定的一种新的结合因子。 其结合 UV、顺铂或变性损伤的DNA;在XP组E中不存在 在顺铂耐药细胞中表达水平增加 DNA修复能力增强 本提案的目的是了解 通过以下方式更充分地检测XP中的分子缺陷： 1. XPE-BF的表征。 要了解XPE-BF如何识别 损伤的DNA，结合XPE-BF的病变谱将被定义。 到 探讨XPE-BF在肿瘤治疗中的意义， 将在具有不同转移性的细胞中研究XPE-BF水平的变化。 潜在的，在他莫昔芬和类固醇激素的反应，并在患者 正在接受化疗 2. XPE-BF基因的分离。 该蛋白质现已纯化， 足够大的量以允许切割、肽测序，以及 用于筛选cDNA的寡核苷酸的定义。 cDNA和基因将 进行克隆和表征，以了解XPE-BF如何作为修复 蛋白 3 .第三章。 证明XPE-BF在着色性干皮病中存在缺陷。 XPE-BF将 测试其以下能力：（a）通过显微注射拯救XP E组细胞 和（B）赋予对顺铂和其他药物的抗性 正常细胞中的抗癌药物。 XP E组患者中的突变将是 其特征在于确定这些个体中的分子缺陷。 四、 XPE-BF如何参与DNA修复的研究。 几种方法 将用于与XPE-BF相互作用的理想异源蛋白。 这些蛋白质在其他XP组中可能有缺陷， XP修复途径中的额外步骤。 XPE-BF中的关键域， 将定义DNA结合和蛋白质相互作用。 克隆的cDNA 将用于生产野生型和突变型XPE-BF，用于在 体外修复系统。 总体目标是了解DNA修复的生物化学， 分子水平。 这一奋进的成功不仅会产生影响， 对于XP患者来说，也是为了了解突变和癌症， 所有人类的易感性。

The inherited disease xeroderma pigmentosum (XP) is characterized by severe sensitivity to ultraviolet (UV) radiation and a high incidence of skin cancer. The biochemical defect is in a pathway for the excision repair of DNA damage produced by UV, the antitumor drug cisplatin, and many other agents that produce bulky DNA adducts. This DNA repair pathway involves multiple proteins, since there are at least seven genetic complementation groups, A through G. An early step in repair must involve recognition of the site of DNA damage. Recently, a candidate protein for this function, XPE binding factor (XPE-BF), was identified in this laboratory. It binds to DNA damaged by UV, cisplatin, or denaturation; is absent in XP group E cells; and is expressed at increased levels in cisplatin resistant cells with increased DNA repair. The aims of this proposal are to understand the molecular defect(s) in XP more fully by: 1. Characterization o f XPE-BF. To understand how XPE-BF recognizes damaged DNA, the spectrum of lesions that bind XPE-BF will be defined. To investigate the significance of XPE-BF in cancer treatment, be modulation of XPE-BF levels will be studied in cells with differing metastatic potential, in response to tamoxifen and steroid hormones, and in patients undergoing chemotherapy. 2. Isolation of the gene for XPE-BF. The protein has now been purified in quantities large enough to permit cleavage, peptide sequencing, and the definition of oligonucleotides for screening cDNA. The cDNA and gene will be cloned and characterized to understand how XPE-BF acts as a repair protein. 3 . Proof that XPE-BF is defective in xeroderma pigmentosum. XPE-BF will be tested for its ability to: (a) rescue XP group E cells by microinjection of purified protein, and (b) confer resistance to cisplatin and other anticancer drugs in normal cells. Mutations in XP group E patients will be characterized to define the molecular defect in those individuals. 4 . The study of how XPE-BF participates in DNA repair. Several methods will be used to ideally heterologous proteins that interact with XPE-BF. Such proteins may be defective in other XP groups and will define additional steps in the XP repair pathway. Critical domains in XPE-BF for both DNA binding and protein interactions will be defined. The cloned cDNA will be used to produce both wild type and mutated XPE-BF for testing in an in vitro repair system. The overall goal is to understand the biochemistry of DNA repair at the molecular level. Success in this endeavor will have implications not only for XP patients, but also for understanding mutagenesis and cancer susceptibility in all humans.